Design and synthesis of multi-functional small-molecule based inhibitors of amyloid-β aggregation: Molecular modeling and in vitro evaluation

Amyloid-β1–42 (Aβ42) peptide aggregate formation in the brain plays a crucial role in the onset and progression of Alzheimer’s disease. According to published research, the Aβ monomer’s amino acid residues KLVFF (16–20) self-associate to create antiparallel β-sheet fibrils. Small compounds can prevent self-assembly and destroy Aβ fibrils by attaching to the Aβ16–20 regions of Aβ42. To enhance biological characteristics and binding affinity to the amyloid beta peptide, β-sheet breaker small molecules can be developed and modified with various scaffolds. In the current study, a novel series of 2,3-disubstitutedbenzofuran derivatives was designed and created by fusing the benzofuran core of a known iron chelator, neuroprotective, and neurorestorative agent, like VK-28, with a motif found in the structure of a known muscarinic inhibitor and amyloid binding agent, like SKF-64346. Measurements of the binding affinity and in vitro aggregation inhibition of the Aβ42 peptide were made using the thioflavin T (ThT) test. Using AutoDock 4.2 software, molecular docking studies of the synthesized compounds were performed on the monomer and fibril structures of amyloid beta peptide. The compounds 8a–8g exhibited strong binding energy and affinity to Aβ fibrils as well as a 50%–67% reduction of the growth of Aβ aggregation. Finally, the positive traits of our recently synthesized compounds make them excellent candidates for additional in vivo testing as a "β-sheet breaking agent."


Introduction
Alzheimer's disease (AD) is a degenerative brain illness that impairs memory, reasoning, and reasoning skills, making daily tasks difficult. Since 1910, when the name AD was first coined, it has remained a hot topic and a challenging condition to understand for experts all around the world [1]. According to WHO figures, almost 55 million people worldwide suffer from dementia, with over 60% of them living in low-and middle-income countries. As the population of elderly people develops in almost every country, this number is expected to rise to 78 hybrid chemicals based on this new paradigm, such as benzofuran and acridine derivatives (Fig 2). The benzofuran or acridine motif inhibits Aβ-aggregation in these hybrid drugs, while the alkylamine moiety inhibits cholinesterase (AChE and BuChE) [19][20][21][22]. A group of researchers revealed, in 1996, that acetylcholine esterase is prone to Aβ aggregation, with the peripheral anionic site playing a key role (PAS). Cell adhesion, neurite outgrowth, and amyloid deposition are all regulated by the PAS [23]. Several compounds (Fig 2) were designed to interact with both the central and PAS systems in order to have antiaggregant and anticholinesterase properties [24]. In addition, the existence of an iron-responsive region in the APP transcript's 5'-UTR [25] revealed a strong relationship between iron metabolism and the AD pathogenic process. The development of iron-chelating neuroprotective neurorescue therapy is being combined with multi-modal tasks that act on several brain regions in recent emerging approaches. M30, HLA-20 (8-hydroxyquinoline derivatives) [26,27], and quercetin analogues (polyphenol flavonoids) (Fig 2) showed the most action in terms of iron chelation, radical scavenging, and inhibiting iron-catalyzed lipid membrane peroxidation [28]. The ability of polyphenols to prevent Aβ aggregation has been attributed to the combined action of the phenolic moieties, which can either stack or interact hydrophobically with Aβ aromatic residues and insert into the space of Aβ aggregates, and/or interact via hydrogen bonding through phenolic hydroxyl groups [29]. In light of these findings, researchers considered developing a range of innovative multimodal drugs, such as AChE and Aβ-aggregation dual inhibitors clearly inspired by the SKF-46346 and structure and an iron-chelating neuroprotective neurorescue 8-hydroxyquinoline moiety. In our work we are prompted to synthesize a series of hybrid compounds (Fig 3) consisting of: a-benzofuran motif of SKF-64346, AChE/BChE inhibitors with inhibitor activity of Aβ-fibrill self aggregation. b-a substituted piperazine spacer, tertiary amine helps for cross BBB. c-8-hydroxyquinoline moiety, metal chelator, neuroprotective and neurorestorative. dvarious substitutes on the aromatic ring at position 2 of benzofuran scaffold were included in order to study the substituent effect on the electronic configuration of the whole structure and its effect on the biological activity of the compounds. point apparatus (Stuart Scientific, Stone, Staffordshire, UK), and were uncorrected. Precoated silica gel plates (Kieselgel 0.25 mm, 60G F254, Merck, Darmstadt, Germany) were used for thin layer chromatography (TLC) using chloroform/methanol (8:2) as developing system. All the chemical structure spectral analysis was done at the research center, College of Pharmacy, King Saud University, Saudi Arabia. Infrared (IR) spectra (KBr discs) were performed using a FTIR spectrophotometer (Perkin Elmer, Shelton, CT, USA). Nuclear magnetic resonance (NMR) spectra were obtained using NMR spectrophotometer (Bruker, Flawil, Switzerland) operating at for 1H and 125.76 MHz for 13C. Mass spectra were taken on a model 320 MS spectrometer (Varian, Lexington, KY, USA). Elemental analyses were performed on a model 2400 elemental analyzer (Perkin Elmer). Biological investigation experiment was done at the Stem Cell Therapy Program, King Faisal Specialized Hospital and Research Center.
Synthesis of 5-((piperazin-1-yl)methyl)quinolin-8-ol 5. Piperazine anhydride (4, 1.9 g, 22 mmol) and piperazine dihydrochloride (6.36 g, 40 mmol) were dissolved in 20 ml of ethanol and then added to a three-necked flask equipped with a magnetic stir bar and reflux condenser. The flask was heated at 75˚C for three hours while being vigorously stirred. Then, over the course of 30 minutes, 5-chloromethyl-8-hydroxyquinoline hydrochloride (3, 5.04 g, 22 mmol) solution was dropped gradually into the heated solution and the mixture was refluxed for additional 4 h. The reaction mixture was cooled, and the piperazine dihydrochloride that had precipitated was removed by filtration and washed with ethanol three times. The filtrate and washes were concentrated in vacuo and treated with saturated soda solution. The aqueous layer of crude resulting in 5-((piperazin-1-yl)methyl)quinolin-8-ol hydrochloride (5) was extracted into CH 2 Cl 2 (3 × 15 ml). The combined organic extracts were dried over Na2SO4 anhydrous and were concentrated in vacuo. The obtained oily product was purified by flash column chromatography on silica gel (1:3 MeOH-CH 2 Cl 2 ) to produce 5.0 g. (83%) of 5-((piperazin-1-yl)methyl)quinolin-8-ol (5).

Thioflavin T (ThT) assay to study Aβ aggregation
The SensoLyte1 Thioflavin T ß-Amyloid42 Aggregation Kit was purchased from AnaSpec Inc. (34801 Campus Drive Fremont CA 94555 USA). To prevent self-aggregation, hexafluoroisopropanol (HFIP) at a concentration of 5 mg/ml was first applied to Aβ1-42 peptides. The peptide-containing clear solution was then aliquoted into a micro centrifuge tube. Under a stream of nitrogen, the HFIP was evaporated until a transparent film was all that was left in the test tube. After that, DMSO was used to dissolve the pre-treated Aβ1-42 samples to create a stable stock solution (Aβ1-42 1 mM). The tested substance (25 mM) was either present or absent in a combination containing the peptide (10 mL, 50 mM, final concentration) that was incubated at 37˚C for 48 hours. Using the thioflavin-T fluorescence technique, the production of amyloid fibrils was quantified. Additionally, blank tests using Aβ1-42 with or without inhibitors and 50 mM phosphate buffer (pH 7.4) were run. Following incubation, samples were diluted with 50 mM glycine NaOH buffer (pH 8.0) containing thioflavin-T to create a final volume of 200 mL. (5 mM). Five minutes later, the fluorescence intensities were measured (excitation, 440 nm; emission, 484 nm). The expression (1-IFi/IFc) x 100% was used to compute the percentage of inhibition of aggregation, where IFi and IFc are the fluorescence intensities obtained for Ab in the presence and absence of inhibitors, respectively, after removing background. Three copies of each measurement were made [31,32].

Determination of ligand/iron(III) stoichoimetries using UV/Vis spectrophotometry
Analytical reagent grade ferric chloride was dissolved in 100% ethanol to create a stock solution of ferric chloride (1.35 X 10-4 M). Compound 8e was dissolved in ethanol to create the ligand solution (1.35 X 10-4 M). On a Shimadzu UV-2550 spectrophotometer, UV spectra were captured. The stoichiometric ratios of the iron(III) model systems with compound 8e were calculated using a spectrophotometric technique described by Joe [33]. For a range of iron(III) and ligand mixes, the amount of iron(III)-ligand complex was determined while maintaining a constant volume of 10.0 mL for the entire mixture. The complex's stoichiometric composition was established in light of the results.

ADMET prediction
Using the ChemDraw program (ChemDraw1 Ultra, version 12, 1986-2009, Cambridge Soft Corp., USA), the structures of compounds 8a-g were shown. The import icon in the molecular sketcher was clicked, opening a new window where the produced structure could be selected and subsequently transferred to the SMILES format on the SwissADME server, http://www. swissadme.ch (accessed on 15 April 2020). The "Run" icon was then clicked, supplying the ADMET settings and associated values.

Molecular docking
The 3D model of compounds 8a -8g with the lowest energy levels was chosen for molecular docking studies. The protein data base PDB was initially used to obtain the crystal structures of the human Aβ amyloid monomer (Aβ m) [34] and Aβ amyloid fibril (Aβ f) [35]. The Lamarckian genetic approach molecular docking was performed using AutoDock 4.2 software [36]. The polar hydrogen atoms and Kollman charges of the ligands and Aβ1-42 monomer were identified after the nonpolar hydrogen atoms were mixed using AutoDock tools and saved in.pdbqt format. The ligands were kept flexible and the Aβ1-42 monomer was kept rigid in the grid box during the docking method to establish the ligands' free rotation around it. An AutoGrid grid box covering the entire Aβ1-42 monomer structure was made with dimensions of 126 X 68 X 68 Å, grid spacing 0.375 Å, and coordinate centers X = -1.739, Y = 1.658, and Z = -6.771 in order to compare the binding energies between the synthesized compounds and the Aβ1-42 monomer and to determine the best structural conformation. Ten docked conformations that all achieved structures with a tolerance of two were constructed for root mean square deviation (RMSD), and their docking energies were sorted to provide the findings.

Thioflavin T (ThT) assay to study Aβ aggregation
One of the most widely used benzothiazole derivatives, thioflavin T (ThT), is used as a dye for the in vitro and in vivo detection and quantification of amyloid beta plaques in the brains of AD patients. To do this, fluorescent spectroscopy is used to assess the fluorescence intensity of bound ThT to amyloid fibrils. This colorful molecule is made up of a benzylamine ring and a benzothiazole ring connected by a single carbon-carbon bond. Two rings can smoothly spin across the single carbon-carbon link and quench the fluorescence intensity when this molecule is activated by a photon in a solution. However, when ThT molecules connect noncovalently with the beta sheets of amyloid fibrils, the two rings become stationary on amyloid aggregations and increase their fluorescence intensity [32]. At doses of 1 μM with thioflavin T (20 μM), the synthetic compounds 8a-8g were tested for their in vitro inhibitory action on Aβ-amyloid aggregation. In Fig 4, the fluorescence spectrum is shown. Thioflavin T assay results indicate that compounds 8a-8g can inhibit the growth and development of fibrils by reducing the fluorescence of ThT molecules at a wavelength of around 485 nm (Fig 4A). Curcumin and its analogs had the same effects [37]. According to Eq 1, the proportion of Aβ-amyloid peptide aggregation inhibition was estimated ( Table 2 and Fig 4B and S4 File). The studied compounds demonstrated encouraging levels of Aβ-peptides aggregation inhibition, ranging from 54.12% to 67.52%. The tested compounds explored comparable inhibition percent values with highest values assigned for compound 8a and lowest value assigned to compound 8g.
Where Tested RFU is the relative fluorescence units of samples that contain reaction mixtures and tested compounds; Blank RFU is the relative fluorescence units of a solvent sample (which is devoid of both tested compounds and reaction mixtures); and maximum control RFU is relative the fluorescence unit of samples that contain only reaction mixtures (and not test compounds).

Determination of ligand/iron(III) stoichoimetries using UV/Vis spectrophotometry
The wavelength scan of compound 8e showed a primary absorption at 307 nm. The band at 307 nm provided a bathochromic shift to longer wavelength 322 nm upon additions of Fe(III) ions to constant concentration of the ligand solution. The absorbance at 322 nm is shown against the ligand/Fe(III) molar ratio in Fig 5. The greatest intensity of the band at 322 nm, attributed to the Fe(III) complex following the addition of 3 mol equivalents of Ligand to the Fe(III) solution, suggests that the iron complex generated has a stoichiometry of 1:3 for Fe(III) and Ligand.

ADMET prediction
Molecule characteristics such as hydrophobicity, electronic distribution, hydrogen bonding, molecule weight, pharmacophore entity, bioavailability, reactivity, toxicity, and metabolic stability influence the similarity of a medicine. One of the methods most frequently used to determine a compound's solubility and permeability and, thus, to determine whether or not it is a medication candidate is Lipinski's rule. The Lipinski rule of five indicates that a molecule is more likely to have poor absorption or penetration if it includes more than five H-bond donors, a molecular weight (MWt) more than 500, a log P value above 5, and more than 10 Hbond acceptors. Table 1 illustrates the drug likeness of the compounds 8a-g. Compounds 8ad, 8f values lie within the usual range and their bioavailability scores are 0.55 and they do not  all deviate from the Lipinski rule while compounds 8e, 8g have one violation of lipinski's rule because of its molecular weight values higher than 500. The studied compounds 8a-e are extensively absorbed via the GI after oral administration, are able to cross the blood-brain barrier (BBB), and can be eliminated by P-glycoprotein (P-gp). Compounds 8g, on the other hand, are significantly absorbed through GI but are unable to cross the blood-brain barrier. This might be explained by the fact that compound 8g has a greater molar refractivity (MR) value (MR: 169.41) than the other compounds put to the test. Compounds 8a-g have no Brenk or PAINS descriptor alarms, which is another encouraging sign that they could be therapeutic candidates. In medicinal chemistry, the Brenk and pan assay interference compounds (PAINS) structural warnings have been utilized to anticipate the presence of unstable, reactive, and hazardous fragments.

Molecular docking studies
One of the most used computational techniques, molecular docking can illustrate crucial biochemical processes by forecasting the binding locations of ligands to the target [38]. We therefore performed molecular docking studies using AutoDock 4.2 software [36] to determine the molecular interactions and binding affinity between the compounds 8a-8g and Aβ1-42 monomer (Aβm) and Aβ1-42 fibril (Aβf). The protein database PDB was initially used to obtain the crystal structures of the human Aβ amyloid monomer (Aβ m) [34] and Aβ amyloid fibril (Aβ f) [35]. The synthesized compounds 8a-8g were drawn, optimized, and transferred to pdbqt format before being docked to the structures of the Aβm and Aβf. The docking study's findings matched the experimental Aβ1-42-aggregation inhibition percentage. The docking data produced the preliminary estimation of the free binding energies revealing the effectiveness of the ligand-receptor complexed system (Table 2), binding sites, and the residues involved in interactions with the binders 8a-8g (Table 3 and Figs 6 and 7). The binding free energy score for fibril protein was lower than that for the amyloid monomer, suggesting that these substances can bind to fibrils through positive interaction. For the complexes of compounds 8a-Aβm, 8b-Aβm, 8c-Aβm, 8d-Aβm, 8e-Aβm; 8f-Aβm and 8g-Aβm docking has calculated the binding energy to be -8.79, -7.78, -8.57, -8.35, -9.73, -8.56 and -8.92 kcal/mol, respectively. When the ligands are coupled to Aβm via non-covalent interactions to form stable complexes, the docking analysis predicted the energetically advantageous poses of the ligands in the binding site, the central hydrophobic area (Lys16 to Ala21), of the Aβm (Table 3). In these bound complexes, the development of hydrogen bonds and hydrophobic interactions were significant factors (Fig 6). The acquired docked poses of the ligands in the binding cavity revealed the hydrogen-bonding residues to the ligands, the core hydrophobic area amino acid residues 14-23 of the Aβm that is essential for monomer-monomer interactions, and the aggregation of the Aβ peptide into the Aβ fibril [28,39]. A critical Table 3. Interacting amino acid residues of amyloid monomer with compounds 8a-8g.

Conclusions
The work included the design and synthesis of new small molecules as hybrids of benzofuran and 8-hydroxyquinoline moieties that are linked together through piperazine spacer. The Table 4. Interacting amino acid residues of amyloid fibril with compounds 8a-8g.

No
Binding residues compounds were proposed as potential multi-target anti-Alzheimer's agents that combine the anti-fibrillation activity of benzofuran scaffold, the antimuscarinic activity of piperazine ring and the iron-chelation properties of 8-hydroxyquinoline moiety. The synthesized compounds exhibited a significant reduction of about 54.12%-76.59% against the Aβ42 aggregation using ThT assay that also supposed a good correlation between docking study and experimental methods. In conclusion, the favorable characteristics of our newly synthesized compounds make it promising candidates β-amyloid-fibrillation inhibitors for further development and studies.
Supporting information S1